The determination of protein nitrotyrosine content has become a frequently used technique for the detection of oxidative tissue damage. Protein nitration has been suggested to be a final product of the production of highly reactive nitrogen oxide intermediates (e.g. peroxynitrite) formed in reactions between nitric oxide ( NO) and oxygen-derived species such as superoxide. The enzyme prostaglandin H synthase-2 (PHS-2) forms one or more tyrosyl radicals during its enzymatic catalysis of prostaglandin formation. In the presence of the NO-generator diethylamine nonoate, the electron spin resonance spectrum of the PHS-2-derived tyrosyl radical is replaced by the spectrum of another free radical containing a nitrogen atom inspite of the fact the PHS-2 activity is independent of NO. The magnitude of the nitrogen hyperfine coupling constant in the latter species unambiguously identifies it as an iminoxyl radical, which is likely formed by the oxidation of nitrosotyrosine, a stable product of the addition of NO tyrosyl radical. Addition of the superoxide dismutase did not alter the spectra, indicating that peroxynitrite was not involved. Western blot analysis of PHS-2 after exposure to the NO-generator revealed nitrotyrosine formation. The results provide a mechanism for nitric oxide-dependent tyrosine nitration that does not require formation of more highly reactive nitrogen oxide intermediates such as peroxynitrite or nitrogen dioxide.